It is known that the chemical synthesis of oligonucleotides of DNA fragments is performed efficiently by the phosphoramidite chemistry, and the coupling reaction gives excellent yield on various solid supports (Oligodeoxy nucleotide synthesis, Phosphoramidite Approach, Serge L. Beaucage in Protocols For Oligonucleotide s and Analogs, Synthesis and Properties, Editor, Sudhir Agarwal, Humana Press, 1993). Similarly, excellent protocols have been developed for the synthesis of RNA, and various biologically active tRNA molecules (Oligoribonucleotide synthesis, The Silyl Phosphoramidite Method, Masad J. Damha and Kevin K. Ogilvie in Protocols For Oligonucleotides and Analogs, Synthesis and Properties, Editor, Sudhir Agarwal, Humana Press, 1993).
A large number of such DNA and RNA molecules with biological functionality carry base labile and modified nucleosides which cannot sustain prolonged basic conditions, generally required during deprotection. Thus, e.g., dihydrouridine present in tRNA requires very mild deprotection conditions, otherwise it is completely decomposed and the quality of synthetic tRNA is compromised. (C. Chaix, D. Molko and R. Teoule, Tetrahedron Letters, 30, 1, 711-74, 1989).
In order to develop protecting groups which are ultra mild in nature have been developed in recent past. Thus 2-(acetoxy-methyl)benzoyl (AMB) group which uses potassium carbonate as mild deprotecting group for their removal has been reported (W. H. A. Kuijpers, J. Huskens and C. A. A. Van Boeckel, Tetrahedron Lett., 31, 6729-6732, 1990) & W. H. A. Kuijpers, E. Kuyl-Yeheskiely, J. H. Van Boom and C. A. A. Van Boeckel, Nucl. Acids Res., 21, 3493-3500, 1993). The AMB group seems attractive, but faces many practical problems in actual use. Although FMOC group has been reported in the prior art for the protection of amino function of 2′-deoxycytidine, 2′-deoxy adenosine and 2′-deoxy guanosine and for the corresponding ribonucleosides (H. Heikkila and J. Chattopadhyaya, Acta Chem. Scand. B 37, No. 3, 263-265, 1983), it has attractive properties as n-protecting group; as pointed out by these authors, the FMOC group offers the capability to be cleaved under very mild alkaline deprotection condition, or by bases capable to carry out selective deprotection via B-elimination of FMOC group (scheme 1).
It is therefore not surprising that other attempts to synthesize N-FMOC protected nucleoside and phosphoramidites have been carried out. Reported in the literature (R. K. Gaur, V. Bobde, M. Atreyi and K. C. Gupta, Indian Journal of Chemistry, 29B, 108-112, 1990), is the preparation of 5′-DMT-n-FMOC-dA (structure 1) and 5′-DMT-n-FMOC-dC (structure 2).
However this team could not synthesize 5′-DMT-N-FMOC-dG (Structure 5). Furthermore, they only synthesized p-methoxy phosphoramidites of 5′-DMT-n-FMOC-dC (structure 3) and 5′-DMT-n-FMOC dA (structure 4), and p-methoxy phosphoramidites have been found to have only limited application in oligonucleotide synthesis.
Also, no N-FMOC protected solid supports were reported by this team.

The synthesis of N2-FMOC-5′-DMT-dG (structure 5) has eluded the researchers so far. In addition, no solid supports or the succinates of the N-FMOC-5′-DMT-deoxy bases (dA ad dC) are revealed by these authors. Thus, there is no information in this prior art as to the applicability of DMT-N-6-FMOC-dA-3′-succinyl-support, to validate the concept that the solid support containing N-6-Fmoc-dA solid supports is expected to minimize or dramatically reduce formation of N-1 (i.e. depurination of 3′-dA base products) during oligo synthesis, nor is there information about the quality of the synthesized 2′-deoxy oligonucleotides. The products are therefore required to improve the quality of the terminal 3′-dA containing oligonucleotides.
There seem to have been no further attempts to make 5′-DMT-N-FMOC dG or the ribonucleoside containing N-Fmoc protected synthons for RNA synthesis and the corresponding cyanoethyl phosphoramidites. The present state of the art in this technology relies on cyanoethyl phosphoramidite chemistry in DNA and RNA synthesis.
It was demonstrated by Heikkila and Chattopadhyaya, (Acta Chem. Scand. B 37, No. 3, 263-265, 1983) that deprotection of FMOC protecting group can be carried out under various very mild basic reaction conditions. It is possible to utilize either aq ammonia condition deprotection, which results in nucleophilic displacement of FMOC protecting group, or by a non-nucleophilic base such as triethylamine, which causes B-elimination of FMOC-active hydrogen group (scheme 1).
The FMOC protecting group is very well established in peptide synthesis and one of the preferred reagent for amino group protection of alpha-amino group of amino acids for step wise peptide synthesis (Carpino, L. A., and Han, G. Y., J. Amer. Chem. Soc., 92, 5748, 1970). However, despite the promise of the FMOC group, Heikkila and Chattopadhyaya, who initiated the synthesis of the FMOC deoxy and ribo nucleosides, themselves switched to another N-protecting group, i.e., 2-nitrophenyl sulfenyl (Nps) for the protection of amino function of cytidine, adenosine, guanosine and the corresponding 2′-deoxy ribo nucleosides (structure 6). (J. Heikkila, N. Balgobin and J. Chattopadhyaya, Acta Chem. Scand B 37, 857-864, 1983)
O-Nitrophenylsulfenyl Protecting Group
Scheme 1. Fmoc-B-Elimination Scheme
It is well known that cyanoethyl protecting group for internucleotide phosphate is eliminated by B-elimination mechanism leading to acrylonitrile and phosphodiester oligonucleotides. (scheme 2).
Scheme 2. Elimination of Cyanoethyl Phosphate Group
Schemes 1 & 2 suggest that it is possible to modulate the FMOC protecting group removal conditions from oligonucleotides. In fact, the FMOC as base protecting group can be removed by the process of B-elimination, just like the B-elimination process to remove cyanoethyl group.
This process therefore offers very attractive potential to use ammonia free oligo synthesis. Furthermore, this process has the potential to offer deoxyoligonucleotides for complete deprotection of oligos on solid supports, holdinging great promise for chip based technology. This process and technology have the potential to offer ribonucleotides such as required for chip based technology as well high purity oligonucleotides for microRNA, Si RNA, RNA chips.
The FMOC group, in conjunction with cyanoethyl phosphate protecting group, therefore allows the removal of both FMOC and cyanoethyl groups from the synthesized deoxy and ribo oligonucleotides on the support cleanly, with preferable non aq bases, and on support, all of which properties are useful for many diagnostics applications.
The utility of N-FMOC protected nucleoside has additional significance and importance: When oligo ribonucleotide chimeras comprise mixed bases composed of 2′-fluoro and 2′-ribo bases, they present a challenge in obtaining pure chimera oligonucleotides. It has been well documented by several recent reports that oligonucleotide chimeras having 2′-fluoro-2;′-deoxy bases along with natural ribo bases present a difficulty in obtaining pure oligos. It has been shown that with strongly basic conditions, there is significant loss of fluorine as loss of HF is seen as M−20 peak in Mass spectral analysis. It has also been shown that uracil and cytosine are eliminated to significant extent, when oligo chimeras containing 2′-fluoro-2′-deoxy uridine and 2′-fluoro-2′-deoxy cytidine are part of the chimeras (see scheme 3).

Scheme 3. Graphic Representation of effect on quality of oligonucleotides containing 2′-fluoro nucleosides: (a) deprotection using methyl amine, the usual deprotection condition of protecting groups of bases, (b) loss of fluorine leading to loss of HF, generating significant amount of M−20, (c) loss of cytosine and uracil, the pyrimidine bases is observed quite frequently, d) with the loss of pyrimidine, subsequent cleavage of chain occurs.
The studies as shown in scheme 3 were carried out independently by two groups recently:
Ken Hill, Agilent Technologies, Boulder, Colo.; Identification of Process Related Impurities—Understanding Oligonucleotide Production, TIDES 2007, Las Vegas. Nev. where the author showed depyrimidation of chimera oligonucleotides carrying 2′-fluoro-2′-deoxy pyrimidine in RNA's; and,Nanda D. Sinha, Avecia Biotechnologies Inc., Massachusetts-Depyrimidation, as well as loss of HF and chain cleavage in chimeras having 2′-fluoro-2′-deoxy pyrimidines in RNA sequences. Eurotides, 2005, Munich, Germany.
In order to overcome these difficulties, therefore, it is necessary to develop 2′-fluoro-2′-deoxy nucleosides and corresponding phosphoramidites with the base protecting groups incorporating N-FMOC protecting group, structures for mild deprotection group, preferably via B-elimination pathway so as to maintain integrity of oligo chimeras and attain requisite high quality for therapeutic and diagnostic applications.

Besides the 2′-fluoro nucleosides, 2′-O-alkyl nucleoside phosphoramidites are extensively used in the design of biologically active oligonucleotides for therapeutic and diagnostic applications as fully alkylated or as chimeras. Amongst the 2′-O-alkyl nucleosides and phosphoramidites the most common are 2′-O-Methyl oligonucleotides which have shown enormous promise in drug design and specific diagnostics applications. Thus 2′-Omethyl oligoribonucleotides-RNA complexes have higher Tm than corresponding oligo-deoxy ribonucleosides-RNA duplexes, Iribarren, A. M., Sproat, B. S., Neuner, P., Sulston, I., Ryder, U., and Lamond, A. I., Proc. Natl. Acad. Sci. USA 87, 7747-7751, 1990. Various 2′-OMethyl-N-FMOC protected nucleosides and phosphoramidites offer great advantage in producing high quality of DNA-RNA oligonucleotides and chimera for biological applications.
Defined sequence RNA synthesis is now well established and currently in use for the synthesis and development of vast variety of therapeutic grade RNA aptamers, tRNA's, Si RNA and biologically active RNA molecules. This approach utilizes (as component 1) a ribonucleoside with suitable N-protecting group, generally a 5′-Protecting group, the most popular being dimethoxytriphenyl (DMT) group; a 2′-protecting group, the most popular being t-Butyldimethylsilyl ether; or a 3′-phosphoramidite, the most popular being cyanoethyl diisopropyl (component 1).
This component is then coupled with a nucleoside with a suitable N-protecting group, 2′ or 3′ succinate of a ribonucleoside attached to a solid support. The present invention represents an advance over prior art by achieving this coupling in solid support; the coupling of component 1 and 5′-OH-n-protected-2′,3′-protected-nucleoside are also achieved in solution phase in the presence of an activator to lead to dimers and oligoribonucleotides, followed by oxidation (3→5′ direction synthesis), also lead to protected dinucleoside having a 3′-5′-internucleotide linkage, Ogilvie, K. K., Can. J. Chem., 58, 2686, 1980.
It is by now recognized that the N-FMOC protecting group offers great potential in RNA synthesis of defined sequence.
This group can be utilized in conjunction with various 2′-protecting groups required for RNA synthesis. The most widely utilized 2′-protecting, tert-butyl-dimethylsilyl, which has been extensively developed by Ogilvie and coworkers as 2′-hydroxy protecting group for oligo ribonucleotide synthesis (Ogilvie, K. K., Sadana, K. L, Thompson, E. A., Quilliam, M. A., and Westmore, J. B Tetrahedron Letters, 15, 2861-2864, 1974; Ogilvie, K. K., Beaucage, S. L, Entwistle, D. W., Thompson, E. A., Quilliam, M. A., and Westmore, J. B. J. Carbohydrate Nucleosides Nucleotides, 3, 197-227, 1976; Ogilvie, K. K. Proceedings of the 5th International Round Table on Nucleosides, Nucleotides and Their Biological Applications, Rideout, J. L., Henry, D. W., and Beacham L. M., III, eds., Academic, London, pp. 209-256, 1983).
These studies subsequently have led to continued developments of methods amenable to both solution and solid phase oligonucleotide synthesis, starting with the first chemical synthesis of RNA molecules of the size and character of tRNA (Usman, N., Ogilvie, K. K., Jiang, M.- Y., and Cedergren, R. J. J. Am. Chem. Soc. 109, 7845-7854, 1987; Ogilvie, K. K., Usman, N., Nicoghosian, K, and Cedergren, R. J. Proc. Natl. Acad. Sci. USA, 85, 5764-5768, 1988; Bratty, J., Wu, T., Nicoghosian, K., Ogilvie, K. K., Perrault, J.- P., Keith, G. and Cedergren, R., FEBS Lett. 269, 60-64, 1990). The literature has been comprehensively reviewed in: Gait, M. J., Pritchard, C. and Slim, G., Oligonucleotides and Their Analogs: A Practical Approach (Gait, M. J., ed.), Oxford University Press Oxford, England, pp 25-48, 1991.
Several other protecting groups have been lately employed for RNA synthesis. These include: bis(2-acetoxyethyl-oxy)methyl (ACE), Scaringe, S. A., Wincott, F. E., Caruthers, M. H., J. Am. Chem. Soc., 120: 11820-11821, 1998; triisopropylsilyloxy methyl (TOM), Pitsch, S., Weiss, P. A., Jenny, L., Stutz, A., Wu, X., Hely. Chim. Acta. 84, 3773-3795, 2001; and t-butyldithiomethyl (DTM) (structure 16), Semenyuk, A., Foldesi, A., Johansson, T., Estmer-Nilsson, C., Blomgren, P., Brannvall, M., Kirsebom, L. A., Kwiatkowski, M., J. Am. Chem. Soc., 128: 12356-12357, 2006.
A more recent invention by the scientists at ChemGenes Corporation developed a patent-pending method for the synthesis of RNA in the reverse direction (i.e., 5′→3′direction) for incorporation of many ligands and chromophores conveniently and efficiently at the 3′-end of the RNA molecules. With this approach, appropriate N-FMOC protected nucleosides, deoxy and ribo may be synthesized utilizing the many advantages of “reverse direction synthesis” of DNA and RNA some of which are briefly mentioned at the end of this section.
A novel 2′-protecting group, acetal levulinyl ester (ALE) (structure 15) has been recently proposed (J. G. Lackey and M. J. Damha, Nucleic Acids Symposium Series, No. 52, 35-36, 2008). Similar to this protecting group another 2′-labile protecting group based on similar chemical nature, 2′-O-acetal ester, pivaloyloxy methyl which has been found mild 2′-O protecting group, T. Layergne, A. Martin, F. Debart, J- J Vasseur, Nucleic Acids Symposium Series No. 52, 51-52, 2008. The base protecting group used by these authors was however n-acetyl and tBpac. However N-Fmoc would be an ideal group for deprotection under mild basic or non basic conditions such as a tertiary amine.

Chemically modified RNA has been synthesized having modified arabino sugars, 2′-deoxy-2′-fluoro-beta-D_arabinonucleic acid (FANA; structure 17) and 2′-deoxy-4′-thio-2′-fluoro-beta-D_arabinonucleic acid (4′-Thio-FANA; structure 18) into sequences for SiRNA activities (Dowler, T., Bergeron, D., Tedeschi, Anna-Lisa, Paquet, L., Ferrari, N., Damha, M. J., Nucl. Acids Res., 34, 1669-1675, 2006). Amongst several new 2′-protecting groups which have been developed, the 2′-protecting 2-cyanoethoxymethyl (CEM) (structure 19) has been shown to produce very long RNA by carrying out RNA synthesis in the conventional (3′→5′) direction. However, the quality of these long RNA's remains in question.

But the N-FMOC protected nucleosides having the 2′-protecting group discussed above can be combined and utilized for high purity RNA synthesis. The N-FMOC protecting group offers great potential in RNA synthesis of defined sequence. This includes RNA synthesis in the conventional direction (3′→5′ direction as well as using newly discovered 5′→3′ direction, i.e., reverse direction synthons; structures 20, 21 & 22).
Structures of Reverse Phosphoramidites and Solid Supports:

Chemical synthesis of RNA is desirable because it avoids the inefficiencies and limitation of scale of synthesis such as those introduced by in vitro transcription by T7 RNA polymerase (Helm, M., Brule, H., Giege, R., Florence, C., RNA, 5:618-621, 1999). Chemical synthesis of RNA is particularly desirable for studies of RNA structure and function, and many useful modifications can be achieved selectively, such as site specific introduction of functional groups, viz., disulphide cross linking as a probe of RNA tertiary structures (Maglott, E. J., Glick, G. D., Nucl. Acids Res., 26: 1301-1308, 1999).
Synthesis of long RNA is very important for biologically active molecules such as tRNA, and such synthesis have been achieved (Persson, T., Kutzke, U., Busch, S., Held, R., Harmann, R. K., Bioorgan. Med. Chem., 9:51-56, 2001; Oglvie, K. K., Usman, N., Nicoghosian, K., Cedrgren, R. J., Proc. Natl. Acad. Sci., USA, 85:5764-5768, 1988; Bratty, J., Wu, T., Nicoghosian, K., Ogilvie, K. K., Perreault, J.- P., Keith, G., Cedergren, R. J., F.E.B.S. Lett., 269:60-64, 1990; Gasparutto, D., Livache, T., Bazin, H., Duplaa, A. M., Guy, A., Khorlin, A., Molko, D., Roget, A., Teoule, R., Nucl. Acids. Res., 20:5159-5166, 1992; Goodwin, J. T., Stanick, W. A., Glick, G. D., J. Org. Chem., 59:7941-7943, 1994).
The aforementioned techniques of RNA synthesis in reverse direction (5′→3′ direction) make the introduction of a number of groups required for selective introduction at 3′-end practical and convenient. The experimental data at ChemGenes Corp. showed higher coupling efficiency per step during automated oligo synthesis with reverse RNA amidites (structures 21, 22, 23), and therefore greater ability to achieve higher purity and to produce very long oligos. It was also demonstrated that the process of the present invention leads to oligonucleotides free of M+1 species, which commonly lead to closer impurities as shoulder near desired peak during HPLC analysis or purification or Gel purification. Such reverse RNA structures replacing the standard n-protecting group with n-FMOC protecting group offer additional unforeseen advantages.